Estriol therapy for multiple sclerosis and other autoimmune diseases

ABSTRACT

The present invention discloses administering steroid hormones to mammals to treat autoimmune related diseases, more particularly, Th1-mediated (cell-mediated) autoimmune diseases including: multiple sclerosis (MS), rheumatoid arthritis (RA), autoimmune thyroiditis and uveitis. Most preferably the invention is used to treat a patient with a therapeutically effective amount of estriol of 8 milligrams once daily via oral administration to treat the symptoms or prevent the onset of multiple sclerosis.

This application is a continuation of U.S. patent application Ser. No.13/763,814, filed Feb. 11, 2013, which is a continuation of U.S. patentapplication Ser. No. 11/151,040, filed Jun. 13, 2005, now U.S. Pat. No.8,372,826, which is a continuation of U.S. patent application Ser. No.10/131,834, filed Apr. 24, 2002, now U.S. Pat. No. 6,936,599, whichclaims priority to U.S. Provisional Patent Application No. 60/286,842,filed Apr. 25, 2001, each of which is hereby incorporated by referencein its entirety.

This invention was made with government support under Grant No. NS036680 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to steroidal therapies for treatingautoimmune diseases and, more particularly, to administering primaryagents being estrogens or estrogen receptor active agents for thetreatment of cell mediated diseases. Optionally, secondary agents whicheffect the immune system may also be co-administered. Finally, treatmentkits are provided containing at least one primary agent and at least onesecondary agent for treating a patient presenting with symptomology ofan autoimmune disease.

2. General Background and State of the Art

There is a distinct female preponderance of autoimmune diseases duringthe reproductive ages including multiple sclerosis (MS), rheumatoidarthritis (RA), uveitis, myesthenia gravis (MG), Sjogren's syndrome, andHashimoto's thyroiditis.

For example, MS is a chronic, and often debilitating disease affectingthe central nervous system (brain and spinal cord). MS affects more than1 million people worldwide and is the most common neurological diseaseamong young adults, particularly woman. The exact cause of MS is stillunknown. MS attacks the nervous system resulting in myelin sheathssurrounding neuronal axons to be destroyed. This demyelinization cancause weakness, impaired vision, loss of balance, and poor musclecoordination. MS can have different patterns, sometimes leaving patientsrelatively well after episodes of acute worsening, sometimes leading toprogressive disability that persists after episodes of worsening. In theworst cases the disease can lead to paralysis or blindness.

Steroid hormones or sex-linked gene inheritance may be responsible forthe enhanced susceptibility of women to these autoimmune diseases. Arole for steroid hormones in susceptibility to autoimmune disease issupported by observations of alternations in disease symptomotology,with alterations in sex hormone levels such as during pregnancy,menopause or exogenous hormone administration (in the form of hormonereplacement (HRT) or oral contraceptives (ORC)). For example, women withMS and RA have been reported to experience remission of symptoms duringlate gestation. Particularly, MS patients have been reported to show adecrease in relapse rate in pregnancy.

Normally, cell-mediated immunity is mediated by T helper cell (Th1)secretion of interferon gamma (IFN-γ) and tumor necrosis factor beta(TNF-b). In contrast, humoral immunity is mediated by another group of Thelper cells (Th2) secreting interleukin (IL)-10, lL-4, IL-5 and IL-6. Asystemic shift toward humoral immunity (or Th2-mediated immunity) hasbeen noted during pregnancy. During pregnancy, cell-mediated immunity isdecreased and humoral-mediated immunity is increased thereby promotingfetal survival. Thus, this systemic shift in the immune system mayexplain why cell-mediated diseases, including MS and RA have beenreported to improve during pregnancy.

Although a shift toward humoral-mediated immunity has been demonstratedduring human pregnancy, mechanisms which induce this shift remainunclear. One possibility is local production of Th2 (or humoralmediated) cytokines by the placenta. Another possibility is theproduction of Th2 cytokines by immune cells, consequent to changedlevels of steroid hormones during pregnancy. Consistent with the latterpossibility, in vitro studies have demonstrated the ability of thesteroid progesterone to increase IL-4 production and the ability of thesteroid 17β-estradiol to increase IL-10 production during T-lymphocyteresponses. However, it remains unclear what cellular mechanisms areinvolved in regulating in vivo amelioration of autoimmune symptomology.

Examples of potential candidates which effect may effect MS duringpregnancy include: Sex hormones (estrogens, progesterone), cortisol,vitamin D, alpha-fetoprotein, human chorionic gonadotropin and pregnancyspecific glycoproteins.

Further, some studies have suggested that a unique pregnancy factortermed “early pregnancy factor” is responsible for improved progressionof cell-mediated autoimmune diseases during pregnancy. Other studieshave suggested a role for microchimerism. Still others suggest a rolefor local factors such as TGF β or estriol (E3) which is known to beproduced by the placenta during pregnancy. Of note, E3 is at its highestserum levels in the third trimester of pregnancy. However, E3's role inameliorating symptoms of autoimmune diseases in humans is unclear.

Studies in laboratory animals have established that experimentalautoimmune encephalomyelitis (EAE) and other Th1 (cell-mediated)autoimmune diseases in mice improve during pregnancy.

Specifically, treatment with late pregnancy levels of estriol orsupraphysiological doses of estradiol (5 times pregnancy levels) wereshown to delay the onset of clinical EAE after disease wasexperimentally induced by immunization of mice (Jansson et al. 1994).However, there was no investigation as to how estrogens delayed the dayof onset of disease, nor as to whether disease severity was effected inthese animals once symptomology occurred.

In another study, it was shown that EAE disease severity could bereduced by treatment with estriol, either before or after disease onset.Treatment of EAE mice with 90 day release pellets of 5 milligrams or 15milligrams of estriol (E3) was shown not only to decrease diseaseseverity but also to enhance autoantigen specific humoral-immunity,increase production of the Th2 cytokine IL-10 and reduced inflammationand demyelination in EAE mice. Importantly, these changes in the diseasewere induced by a dose (5 mg) which was shown to yield estriol levels inserum that were similar to those which occur during late pregnancy (Kimet al., Neurology, 50(4 Supp. 4):A242-245, April 1998, FASEB Journal12(4):A616, March 1998 and Neurology 52(6):1230-1238, April 1999; hereinincorporated by reference). Thus, these results suggested that steroidhormones, and estriol in particular, may be involved in the ameliorationof autoimmune reactions in the EAE animal model.

Other groups later demonstrated that estrogen potentiated the effects oftreatment with TCR proteins to reduce autoimmune reactions in EAE mice.Offner. et al. FASEB Journal 14(6):A1246, April 2000; Int. Journal ofMol. Medicine 6 (Supp. 1): SS8, October 2000 and Journal of Clin.Invest. 105(10):1465-1472, May 2000). Further, it was shown in animalstudies that estrogen suppressed the onset EAE in mice (Ito. et al.Journal of Immunology, 167(1): 452-52, 2001) and that presumed diestruslevels of estrogens reduced some manifestations of active EAE in mice.Bebo et al. Journal of Immunology 166(3): 2080-9, 2001.

However, the etiology and disease progression of EAE and MS are notidentical, thus it is unclear that estrogens alone would be effective inameliorating autoimmune responses in human patients. Indeed, not only isit unknown whether pregnancy doses of estrogens might be protective inhumans with autoimmune disease, it is unclear even in mice whether lowdoses of estrogens are protective. For example, it has been reported bysome that ovariectomy of female mice makes EAE disease worse (Matejuk etal., 2001), while others have found that ovariectomy had no effect ondisease severity (Kim et al., 2001: Voskuhl and Palaszynski, 2001a;Voskuhl and Palaszynski. 2001b). Thus, it is controversial whether lowlevels of estrogens, as they exist during the menstrual cycle, areprotective even in mice.

Data from human studies to date have shown no clear benefit of steroidsin treating any autoimmune disease. In humans, administration ofavailable hormone therapies (including HRTs and OCPs) containing amixture of sex hormones cause some autoimmune diseases to improve whileothers worsen.

For example, there has been no conclusive evidence that women areprotected from or have a decrease in symptomology or relapse rates dueto sex steroids. One study noted that past use of oral contraceptives inhealthy women had no effect on subsequent risk to develop MS (Hernan etal. 2000). Further, another study found that the incidence rates for MSin current users were not decreased as compared to never-users(Thorogood and Hannaford, 1998). Thus, low dose of the estrogens in oralcontraceptives are not of sufficient type or dose to ameliorate theimmunopathogenesis of MS even temporarily during intercurrent use. Atbest, in one study, patients had the subjective impression thatpre-existing MS symptoms (as opposed to relapse rates) worsen during thepremenstrual period and that the use of oral contraceptives may havedecreased this worsening (Zorgdrager and De Keyser, 1997). Importantly,the lack of reports of an effect of oral contraceptive therapy on MSrelapses is in marked contrast to what has been observed duringpregnancy

In contrast, it has been shown that women had a lower the risk ofdeveloping MS during pregnancy compared to non-pregnant states(Runmarker and Andersen, 1995). Due to the numerous changes that occurduring pregnancy, hormonal and nonhormonal (as listed above), theetiology of the beneficial effect of pregnancy may or may be related tosex steroid fluctuations. It has also been reported for decades thatpregnancy decreases MS relapses (Abramsky, 1994; Birk et al. 1990; Birket al. 1998; Damek and Shuster, 1997; Runmarker and Andersen, 1995;Confavreux et al., 1998). These studies have shown that the latter partof pregnancy is associated with a significant reduction in relapses,while there is a rebound increase in relapses post partum. In contrast,the absence of such an effect on relapses during OCP or HRT indicatethat low level sex steroids are not adequate to treat these symptoms.

Further, women having rheumatoid arthritis that were treated with HRTdid not show significant improvement in their symptomology. DaSilva andHall, Baillieres Clinical Rheumatology 1992, 6:196-219; Bijlsma at al.Journal of Repro. lmm. 28(3-4):231-4, 1992; Hall et al. Annals of theRheumatic Diseases, 53(2): 112-6, 1994.

Thus, the low doses of hormones found naturally during the menstrualcycle or in ORT and HRT have not been shown to be effective atameliorating the symptomology of autoimmune diseases. This is in spiteof the observation that women having MS have a decreased relapse rateduring late pregnancy. Thus, a challenge has been to identify a hormoneand a treatment dose that is therapeutic in treating particularautoimmune diseases, while minimizing undesirable side effects.Obviously, the dose and method of administration of steroids in humansdiffers from steroid treatment in laboratory animals due to toxiceffects of prolonged exposure by patients to steroid hormones. Inparticular, there are clinical concerns of inducing breast orendometrial cancers in women requiring long term exposure to steroidhormones.

INVENTION SUMMARY

A general object of the present invention is to provide a method ofadministering steroid hormones to mammals to treat autoimmune relateddiseases, more particularly, Th1-mediated (cell-mediated) autoimmunediseases including: multiple sclerosis (MS), rheumatoid arthritis (RA),autoimmune thyroiditis. uveitis and other autoimmune diseases in whichclinical symptomology has shown improvement during the third term ofpregnancy.

In accordance with one aspect of the present invention, these objectivesare accomplished by providing a treatment for autoimmune relateddiseases with a selected dose and course of a primary agent being anestrogen or estrogen receptor-effective composition.

In accordance with one aspect of the present invention, these objectivesare accomplished by providing a patient with a therapeutically effectiveamount of estriol, comprising from about 4 to 16 milligrams per day, ormore specifically, about 8 milligrams once daily via oraladministration.

In accordance with another aspect of the present invention, theseobjectives are accomplished by providing a therapeutically effectiveamount of a primary agent in combination with a therapeuticallyeffective amount of a secondary active agent, such as progesterone,glucocorticoids and/or known or experimental drugs used to treatautoimmune diseases.

The above described and many other features and attendant advantages ofthe present invention will become apparent from a consideration of thefollowing detailed description when considered in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a schematic depicting the trial design described in Example1; FIG. 1 b is a bar graph depicting human serum levels duringpregnancy, estriol treatment (Tx), and pretreatment (Pre Tx levels).

FIG. 2 a is a bar graph describing the Delayed Type Hypersensitivity(DTH) responses to tetanus and to candida; FIG. 2 b is a bar graphdepicting levels of IFNγ between treatment groups.

FIG. 3 a, FIG. 3 b, FIG. 3 c, FIG. 3 d, FIG. 3 e, and FIG. 3 f are bargraphs depicting each patient's gadolinium enhancing lesion volumes onserial cerebral MRIs, which were assessed at each month during thepretreatment, estriol treatment, and post treatment periods.

FIG. 4 is a bar graph depicting mean percent change in PASAT scoresduring treatment with estriol as compared to pretreatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This description is not to be taken in a limiting sense, but is mademerely for the purpose of illustrating the general principles of theinvention. The section titles and overall organization of the presentdetailed description are for the purpose of convenience only and are notintended to limit the present invention.

Generally, the invention involves a method of treating mammal exhibitingclinical symptoms of an autoimmune disease comprising administering aprimary agent at a therapeutically effective dosage in an effectivedosage form at a selected interval. The treatment is aimed at reducingthe symptomology and/or progression of the disease. In the preferredembodiment of the invention, human patients clinically diagnosed with MS(including both relapsing remitting or secondary progressive typepatients) are treated with an oral preparation of 8 milligrams estrioldaily and have ameliorated symptomology.

Amelioration of the autoimmune disease refers to any observablebeneficial effect of the treatment. The beneficial effect can beevidenced by a delayed onset or progression of disease symptomology, areduction in the severity of some or all of the clinical symptoms, or animprovement in the overall health.

For example, patients who have clinical symptoms of an autoimmunedisease often suffer from some or all of the following symptoms:worsening of pre-existing symptoms (such as joint pain in rheumatoidarthritis), the appearance of new symptoms (new joints affected inrheumatoid arthritis) or increased generalized weakness and fatigue. MSpatients in particular suffer from the following symptoms: weakness,numbness, tingling, loss of vision, memory difficulty and extremefatigue. Thus an amelioration of disease in MS would include a reductionin the frequency or severity of onset of weakness, numbness, tingling,loss of vision, memory difficulty and extreme fatigue. On imaging of thebrain (MRI) amelioration of disease would be evidenced by a decrease inthe number or volume of gadolinium enhancing lesions, a stabilization orslowing of the accumulation of 12 lesions and/or a slowing in the rateof atrophy formation. Immunologically, an increase in Th2 cytokines(such as IL-10) and a decrease in Th1 cytokines (such as interferongamma) would be associated with disease amelioration.

Patients may also express criteria indicating they are at risk fordeveloping autoimmune diseases. These patients may be preventativelytreated to delay the onset of clinical symptomology. More specifically,patients who present initially with clinically isolated syndromes (CIS)may be treated using the treatment paradigm outlined in this invention.These patients have had at least one clinical event consistent with MS.but have not met full criteria for MS diagnosis since the definitediagnosis requires more than one clinical event at another time(McDonald et al., 2001). Treatment of the present invention would beadvantageous at least in preventing or delaying the development ofclinically definite MS.

Primary Agent.

The primary agent useful in this invention is a steroid hormone, moreparticularly an estrogen or a steroidal or non-steroidal estrogenreceptor active agent. Most preferably, the primary agent is estriol(estra-1,3,5(10)-triene-3,16,17-triol, E3), such as estriol succinate,estriol dihexanoate, or estriol sulfamate. However, the primary agentmay be precursors or analogs of estriol, estrone (E1) or precursors oranalogs of estrone, or 17β-estradiol (E2) or precursors (includingaromatizable testosterone) or analogs of 170-estradiol.

The primary agent may also be a metabolite or derivatives of E1. E2 orE3 which are active at the estrogen receptor a or J3. Metabolites andderivatives may have a similar core structure to E1, E2 or E3 but mayhave one or more different groups (ex. hydroxyl, ketone, halide, etc.)at one or more ring positions. Synthetic steroids which are effective atestrogen receptor are also useful in this invention, such as thosedescribed in WO 97/08188 or U.S. Pat. No. 6,043,236 to Brattsand.

The primary agent may also be an estrogen receptor α or β agonistsand/or antagonist. These agonists or antagonists may be steroidal ornon-steroidal agents which bind to and/or cause a change in activity orbinding of at least one of the estrogen receptor α or β subtypes. Forexample, specific agonists of ER alpha and ER beta may be useful in thisinvention (Fritzmeier, et al.). Doses of these agonists may be titratedto achieve an effect on disease similar to that which is observed duringpregnancy and during treatment with pregnancy doses of estriol bymethodologies known to those skilled in the art of steroid pharmacology.

Any one or combination of these estrogens or estrogen receptor activeagents may be used to treat the selected autoimmune disease. Theselection of the estrogens or estrogen receptor active agents can bemade considering secondary side effects of the treatment to the patient.For example, estriol may be selected over 17β-estradiol, because estriolcauses minimal endometrial proliferation and is not associated withincreased risk of breast cancer. Minimal endometrial proliferation isobserved when the long-acting estriol derivative, nyestriol is used.Indeed, because estriol has partial antagonist action on the binding of17β-estradiol to the estrogen receptor in vivo, estriol was at one pointin the past considered as a therapeutic agent for treatment andprevention of breast cancer.

Therapeutically Effective Dosage of the Primary Agent.

A therapeutically effective dose of the primary agent is one sufficientto raise the serum concentration above basal levels, and preferably topregnancy levels or above pregnancy levels. Most preferably, thetherapeutically effective dosage of the primary agent is selected toresult in serum levels in a patient equivalent to the steroid hormonelevel of that agent in women in the second or third trimester ofpregnancy.

For example, during the normal female menstrual cycle estradiol levelsare in the range of about 350 pg/ml serum. During pregnancy, there isabout a 100 fold increase in the level of estradiol to about 10,000 toabout 35,000 pg/ml serum. Correale, et al. Journal of Immunology161:3365 (1998) and Gilmore, et al. Journal of Immunology 158:446. Incontrast, estriol levels are undetectable during the menstrual cycle inthe non-pregnant state. Estradiol levels rise progressively duringpregnancy to levels from 3,000 to 30,000 pg/ml (3 to 30 ng/ml)(www.il-st-acad-sci.org/steroid1.html#se3t).

In one embodiment, where the primary agent is estriol, the preferabledose is from about 4 to 16 milligrams daily, and more specifically,about 8 milligrams daily. In this embodiment, blood serum levelspreferably reach at least about 2 ng/ml, may reach about 10 to about 35ng/ml, or most preferably about 20-30 ng/ml. Sicotte et al. Neurology56:A75. In some embodiments, estradiol (E2) levels would preferablyreach at least about 2 ng/ml and most preferably about to 10-35 ng/ml.In some embodiments, estrone (E1) levels would preferably reach at leastabout 2 ng/ml and most preferably about 5-18 ng/ml (DeGroot and Jameson,1994).

The dosage of the primary agent may be selected for an individualpatient depending upon the route of administration, severity of disease,age and weight of the patient, other medications the patient is takingand other factors normally considered by the attending physician, whendetermining the individual regimen and dosage level as the mostappropriate for a particular patient.

The use of this group of primary agents is advantageous in at least thatother known or experimental treatments for cellular mediated autoimmunediseases are chemotherapeutic immunosuppressants which have significantrisks and side effects to patients, including decreasing the ability ofthe patient to fight infections, inducing liver or heart toxicity whichare not caused by estrogen treatment. Other agents used in MS do notcause these side effect, but are associated with flu-like symptoms orchest tightness. Further, these previously used agents are associatedwith local skin reactions since they entail injections at frequenciesranging from daily to once per week.

Dosage Form.

The therapeutically effective dose of the primary agent included in thedosage form is selected at least by considering the type of primaryagent selected and the mode of administration. The dosage form mayinclude the active primary agent in combination with other inertingredients, including adjutants and pharmaceutically acceptablecarriers for the facilitation of dosage to the patient as known to thoseskilled in the pharmaceutical arts. The dosage form may be any formsuitable to cause the primary agent to enter into the tissues of thepatient.

In one embodiment, the dosage form of the primary agent is an oralpreparation (liquid, tablet, capsule, caplet or the like) which whenconsumed results in elevated serum estrogen levels. The oral preparationmay comprise conventional carriers including dilutents, binders, timerelease agents, lubricants and disintigrants.

In other embodiments of the invention, the dosage form may be providedin a topical preparation (lotion, creme ointment or the like) fortransdermal application. Alternatively, the dosage form may be providedin a suppository or the like for transvaginal or transrectalapplication.

That estrogens or estrogen receptor active agents can be delivered viathese dosage forms is advantageous in that currently availabletherapies, for MS for example, are all injectables which areinconvenient for the user and lead to decreased patient compliance withthe treatment. Non-injectable dosage forms are further advantageous overcurrent injectable treatments which often cause side effects in patientsincluding flu-like symptoms (particularly, β interferon) and injectionsite reactions which may lead to lipotrophy (particularly, glatirameracetate copolymer-1).

However, in additional embodiments, the dosage form may also allow forpreparations to be applied subcutaneously, intravenously,intramuscularly or via the respiratory system.

Secondary Active Agents.

Any one or a combination of secondary active agents may be included inthe dosage form with the primary agent. Alternatively, any one or acombination of secondary active agents may be administered independentlyof the primary agent, but concurrent in time such that the patient isexposed to at least two agents for the treatment of their immunologicaldisease.

The secondary agents are preferably immunotherapeutic agents, which actsynergistically with the primary agent to diminish the symptomology ofthe autoimmune disease. Secondary active agents may be selected toenhance the effect of the estrogen or estrogen receptor active agent,reduce the effect of the estrogen or estrogen receptor active agent oreffect a different system than that effected by the estrogen or estrogenreceptor active agent.

Secondary active agents include immunotherapeutic agents which cause achange in the activity or function of the immune system.

In one embodiment, a secondary agent may be a therapeutically effectiveamount of progesterone, precursor, analog or progesterone receptoragonist or antagonist. Most preferably, the secondary agent is 100-200milligrams of progesterone administered daily. Progesterone incombination with estrogen or estrogen receptor active agent treatment isadvantageous in at least protecting patients against risks associatedwith long term estrogen exposure, including, but not limited toendometrial proliferation and breast cancers.

In another embodiment, a secondary agent may be a therapeuticallyeffective amount of glucocorticoid, precursor. analog or glucocorticoidreceptor agonist or antagonist. For example, prednisone may beadministered, most preferably in the dosage range of about 5-60milligrams per day. Also, methyl prednisone (Solumedrol) may beadministered, most preferably in the dosage range of about 1-2milligrams per day. Glucocorticoids are currently used to treat relapseepisodes in MS patients, and symptomatic RA within this dosage range.

In other embodiments, a secondary agent may be selected from the groupimmunotherapeutic compounds. For example, as β-interferon (Avonex®(interferon-beta 1a), Rebiff® (by Serono); Biogen, Betaseron®(interferon-beta 1b) Berlex, Schering), glatiramer acetate copolymer-1(Copaxone®; Teva), antineoplastics (such as mitoxantrone; Novatrone®Lederle Labs), human monoclonal antibodies (such as natalizumab;Antegren® Elan Corp. and Biogen Inc.), immonusuppressants (such asmycophenolate mofetil; CellCept® Hoffman-LaRoche Inc.), paclitaxel(Taxol®; Bristol-Meyers Oncology), cyclosporine (such as cyclosporin A),corticosteroids (glucocorticoids, such as prednisone and methylprednisone), azathioprine, cyclophosphamide, methotrexate, cladribine,4-aminopyridine and tizanidine.

By way of example, which is consistent with the current therapeutic usesfor these treatments, Avonex® in a dosage of about 0 to about 30 mcg maybe injected intramuscularly once a week. Betaseron® in a dosage of about0 to about 0.25 mg may be injected subcutaneously every other day.Copaxone® in a dosage of about 0 to about 20 mg may be injectedsubcutaneously every day. Finally. Rebiff® may be injected at atherapeutic dose and at an interval to be determined based on clinicaltrial data, However, dosages and method of administration may be alteredto maximize the effect of these therapies in conjunction with estrogentreatment. Dosages may be altered using criteria that are known to thoseskilled in the art of diagnosing and treating autoimmune diseases.

Preferably, secondary agents would be administered in the dosage rangescurrently used to treat patients having autoimmune diseases, includingMS patients. Alternatively, the secondary agents may be administered ata reduced dose or with reduced frequency due to synergistic orduplicative physiological effects with the primary agent.

Preferably, patients exhibiting symptomology of autoimmune diseases aretreated with the above agents (estrogen or estrogen receptor activeagents with or without secondary agents). Most preferably, patientsexhibit autoimmune diseases marked by improvement in symptomology atleast during a treatment regimen, including but not limited to thatreflecting patterns observed during the second or third trimester ofpregnancy.

Kits.

In another aspect of this invention kits are provided for use by thetreating physician in the clinic or prescribed patient forself-administration of treatment. The kits of this invention include atleast one primary agent and one secondary agent in the appropriatedosages and dosage form for the treatment of the patient's clinicalsymptoms.

In a first embodiment of the kit, the primary agent is estriol in dosesof about 4-16 milligrams and the secondary agent is progesterone indoses of about 100 to about 200 milligrams. In a second embodiment ofthis kit, the primary agent is estriol in doses of about 4-16 milligramsand the secondary agent is a glucocorticoid, such as prednisone (about5-60 milligrams per day) or methyl prednisone (1-2 milligrams per day).

In a third embodiment of this invention, the primary agent is estriol indoses of about 4-16 milligrams and the secondary agent is (3-interferonin doses of about 0.25 milligrams of Betaseron® or 30 mcg of Avonex®. Ina fourth alternate embodiment of the kit, the primary agent is estriolin doses of about 4 to about 16 milligrams and the secondary agent isglatiramer acetate copolymer in doses of about 20 milligrams ofCopaxone®.

The kit also preferably contains instructions for use of the kit by theuse by the treating physician or patients to treat their autoimmunedisease. Such information would include at least the schedule for theadministration of the primary agent dose and the secondary agent dose.

Although the present invention has been described in terms of thepreferred embodiment above, numerous modifications and/or additions tothe above-described preferred embodiments would be readily apparent toone skilled in the art.

Example 1 Methods: Trial Design

A crossover design was used with monthly brain MRIs during the six monthpretreatment period, the six month treatment period with oral estriol (8milligrams/day) and the six month post treatment period, with clinicaland laboratory evaluations as demonstrated (FIG. 1A).

Inclusion Criteria.

Women with clinically definite MS, ages 18-50, with an EDSS 0-6.5 whohad been off interferon beta and copolymer-1 for at least six months,and had no steroid treatment for at least three months were eligible. Atleast 5 cm³ of lesion burden on a screening T2 weighted brain MRI wasrequired. Subjects who were pregnant or nursing. on oral contraceptivesor hormone replacement therapy, or who had a history of thrombosis,neoplasm or gynecologic disease, or who had been treated in the pastwith total lymphoid irradiation, monoclonal antibody, T cellvaccination, cladribine or bone marrow transplantation were excluded.

Patients.

Twelve female patients with clinically definite MS were enrolled. Sixhad RR disease and six had SP disease. All six RR and four of six SPpatients completed the entire 18 month study period. One SP patient wasdiscontinued from the study because of prolonged treatment with steroidsfor tonic spasms by an outside neurologist and the other did not wish togo untreated in the post treatment period. Of the ten patients whocompleted the entire study, the mean age was 44 years (range 28 to 50years) and the mean EDSS was 3.3 (range 1.0 to 6.5). The mean EDSS scorefor the SP patients was 5.0 while the mean EDSS for the RR patients was2.2. The 18 month trial was extended in RR patients whereby treatmentwas re-instituted.

Medication.

For the initial treatment phase, micronized, U.S.P. graded estriolpowder (Medisca, Inc., Plattsburg, N.Y.) was put into capsules by UCLAPharmaceutical Services. During the extension re-treatment phase in theRR patients, all but one received a capsule of estriol (8milligrams/day) plus progesterone (100 milligrams/day), while the singleRR patient who had a hysterectomy received only estriol (8milligrams/day) (Women's International Pharmacy, Madison, Wis.).

Clinical and Safety Measures.

Subjects were evaluated using the Kurtzke's Expanded Disability StatusScale (EDSS) by the same neurologist (RV) throughout the study. At eachvisit the study nurse (RK) administered the paced auditory serialaddition test (PASAT) and the 9-hole peg test. Blood was drawn forSMA12, cholesterol panel, blood counts and hormone levels (estriol.estradiol, estrone, LH, FSH, cortisol, progesterone). Estriol levels inserum were determined by ELISA according to manufacturer's instructions(Oxford Biomedical, Oxford, Mich.).

Delayed Type Hypersensitivity Responses (DTN).

DTH to tetanus (Tetanus Toxoid, Wyeth Laboratories, Marietta, Pa.) andcandida (Candin, Allermed Laboratories, San Diego, Calif.) were testedat two timepoints, once in the pretreatment period at study month 3 andonce at the end of the treatment period at study month 12 (FIG. 1 a). Agroup of six untreated healthy control women were also tested twice,spanning the same time interval (9 months). 0.1 ml of each solution wasinjected intradermally on the anterior surface of the forearm.Induration at each injection site was read after 48 hours. Each site wasmeasured twice, once vertically and once horizontally with the averagerecorded. The same nurse (RK) administered all injections and read allresponses on all subjects at both time points.

Reverse Transcription and Polymerase Chain Reaction.

Peripheral blood mononuclear cells (PBMCs) were isolated fromheparinized venous blood and cryopreserved. PBMCs were thawed inparallel from a given patient during the two pre-treatment timepointsand the two treatment timepoints. Total RNA was isolated, DNA wasremoved and mRNA was reverse transcribed. Both IFN-γ and actin wereamplified from the same cDNA, however, the cDNA was diluted 1:9 prior toamplification for actin. Amplification was done in 1 mM MilligramsCl₂using IFNγ and actin primer sequences (Life Technologies, Rockville.Md.). Complementary DNA was amplified for 35 cycles: 45″ @95° C., 60″@54° C. and 45″ @72° C. PCR products were separated on a 1.5% agarosegel containing ethidium bromide and densitometry performed.

MRIs.

Scans were performed on a 1.5T G.E. scanner. The pulse sequencesobtained were a TI-weighted scan with and without gadolinium (Omniscan0.1 mmol/kg) and a PD/T2 weighted scan. Digitized image data wastransferred to a SGI workstation (Silicon Graphics, Inc) for furtherprocessing. The number and volume of new and total gadolinium enhancinglesions was determined using a semiautomated threshold based technique(Display, Montreal Neurological Institute) by a single experiencedoperator (NS). The operator was blinded as to whether patients had RR orSP disease. To calculate T2 volumes, a custom semiautomated, thresholdbased, seed-growing algorithm was used to determine lesion volume afterskull stripping, rf correction and spatial normalization. All scans werecounted by the same technician who was blinded as to whether patientshad RR or SP disease.

Statistical Analysis.

One sample, paired, t tests were used to ascertain significance ofpercent changes in DTH responses, IFNγ levels and PASAT cognitivetesting scores during treatment as compared to pretreatment. Thenonparametric, Wilcoxon's signed rank test was used for statisticalcomparisons in enhancing lesion numbers and volumes on MRI between thesix month baseline period and each treatment period, post treatmentperiod and re-treatment period.

Results. Estriol Levels and Tolerability.

Serum estriol levels during treatment and re-treatment approximatedthose observed in women who were six months pregnant, but were lowerthan those who were 8.5 months pregnant (FIG. 1B). Consistent withprevious reports, estriol was well tolerated with only menstrual cycleabnormalities. There were no significant alterations in any laboratorymeasures including LH, FSH, cortisol, progesterone, estradiol andestrone.

Immune Responses.

Skin testing to tetanus and candida were performed once in thepretreatment period and once at the end of the treatment period todetermine whether they might be decreased with treatment. DTH responsesto tetanus were significantly, P=0.006, decreased at study month 12,when patients had been on estriol for six months, as compared to DTHresponses at study month 3, the pretreatment baseline (FIG. 2A), DTHresponses to candida were decreased less dramatically and more variably.The significant decrease in DTH responses to tetanus from pretreatment(month 3) to treatment (month 12) was not merely due to repeat testingat nine months since healthy, untreated female controls tested atbaseline, then again after nine months, did not demonstrate asignificant decrease in DTH responses as compared to their baseline.These findings are consistent with an estriol induced down-regulation ofTh1 responses in vivo during treatment.

IFNγ is a signature cytokine for Th1 responses. Therefore, we assessedIFNγ levels by RT-PCR of unstimulated peripheral blood mononuclear cells(PBMCs) derived ex vivo from patients during the pretreatment and thetreatment periods. In the six RR patients, levels of IFNγ were variablydecreased at study month 9 (after three months of estriol treatment) andthen significantly decreased, P=0.003, at study month 12 (after sixmonths of estriol treatment) as compared to baseline pretreatment levels(months 3 and 6) (FIG. 2B). In contrast, there was no decrease in IFNγin the four SP patients. These data are consistent with the concept thatthe immune system of RR patients, as compared to SP patients, may bemore amenable to treatments that aim to decrease Th1 responses. Also,the observation that estriol treatment can alter cytokine production byPMBCs is consistent with reports demonstrating estrogen receptors a andβ in immune tissues and cells.

MRIs.

Based on the protective effect of pregnancy on relapse rates in MSpatients and the association of gadolinium enhancing lesions withrelapses, we hypothesized that estriol treatment would have ananti-inflammatory effect as manifested by decreases in enhancing lesionson serial brain MRIs. Compared to the six month pretreatment baselineperiod, the total volume and number of enhancing lesions for all ten MSpatients (6RR, 4SP) decreased during the treatment period. Thisimprovement in the group as a whole was driven by the beneficial effectof estriol treatment in the RR, not the SP, group (FIGS. 3A and 3B).Therapeutic effects of estriol treatment in the RR group were thereforeexamined in further detail. Within the first three months of treatmentof RR patients, median total enhancing lesion volumes were decreased by79%, P=0.02. and numbers were decreased by 82%. P=0.09 (FIGS. 3C and3D). They remained decreased during the next three months of treatment,with lesion volumes decreased by 82%, P=0.01, and numbers decreased by82%, P=0.02. In the post treatment period, median total enhancing lesionvolumes and numbers became variable in the first three months offtreatment, before returning to near baseline levels in the last threemonths of the post treatment period. During the four month re-treatmentextension phase, enhancing lesion volumes decreased again by 88%,P=0.008, and numbers decreased again, this time by 48%, P=0.04, ascompared to original baseline (FIGS. 3C and 3D). Changes in median newenhancing lesion volumes and numbers followed similar patterns as mediantotal lesion numbers and volumes (FIGS. 3E and 3F).

Median T2 lesion volumes for the whole group were 15.3 cm³ (range6.1-33.8), with no significant differences in median T2 volumes betweenRR and SP groups. Consistent with enhancing lesion data, serial T2lesion volumes revealed that estriol treatment tended to be mostbeneficial in RR patients. In the RR group, median T2 lesion volumesremained stable during the six month treatment period (0% change),increased during the six month post treatment period (7.4% higher). andthen declined in the four month re-treatment extension period (2.0°/alower).

Clinical Measures.

Relapses were few and showed no significant changes during the study. Inthe six RR patients, one relapse occurred during the pretreatmentperiod, one in the treatment period, two in the post treatment periodand none in the re-treatment period. No relapses occurred in SPpatients. EDSS and 9 Hole Peg Test scores showed no significant changesduring the study (Table 1).

TABLE 1 Clinical Measures Pretreatment Estriol Treatment Post Treatment3 mo. 6 mo. 9 mo. 12 mo. 15 mo. 18 mo. EDSS scores 6 RR 2.2 2.0 1.5 1.71.8 1.8 (0.6) (0.5) (0.7) (0.6) (0.6) (.05) 4 SP 5.0 5.0 4.9 5.0 5.1 5.0(0.9) (0.9) (1.0) (0.9) (1.1) (0.8) 9 Hole Peg Test scores 6 RR R 22.221.8 22.5 21.5 21.0 21.4 (2.4) (1.6) (2.3) (1.9) (1.7) (2-4) L 24.8 22.924.3 23.3 23.0 22.7 (3.2) (1.6) (2.5) (2.1) (2.1) (2.3) 4 SP R 26.8 29.930.2 31.7 29.4 34.0 (0.4) (2.4) (1.4) (4.8) (5.2) (8.7) L 23.5 25.6 22.724.8 26.7 25.0 (1.4) (2.5) (1.7) (2.6) (0.7) (1.8)

Interestingly, PASAT cognitive testing scores were significantlyimproved in the RR group. but not in the SP group (FIG. 4). Thisimprovement in PASAT scores in RR patients by 14.0% during treatment ascompared to baseline, reached statistical significance, P=0.04. It isunlikely that this improvement was entirely due to a practice effect ofrepeated testing because of the long time interval between testing (9months) and because alternate versions of the test were used in eachpatient. This beneficial effect of estriol treatment on PASAT scores ofRR MS patients is consistent with previous reports describing abeneficial effect of estrogen replacement therapy in surgicallymenopausal women and high dose estrogen treatment in Alzheimer'sdisease. Sicottte, et al. Treatment of Women with Multiple SclerosisUsing Pregnancy Hormone Estradiol: A Pilot Study. Neurology, 56(8 Supp.3):A75, April 2001, and Sicottte, et al. Treatment of Multiple Sclerosiswith the Pregnancy Hormone Estradiol, Submitted to Neurology 2002. areherein incorporated by reference in their entirety.

Example 2

Progesterone in combination with estrogen treatments has been shown toprotect against endometrial proliferation and cancer. Indeed, estrogencannot be given for a lengthy period of time in an “unopposed” fashionin any woman with a uterus. Thus, seven of the 12 patients wanted toremain on estriol after completion of the 18 month study. These patientswere then put back on 8 milligrams of estriol and 100 milligrams ofprogesterone per day. In an extension phase of the study which beganafter completion of the post treatment phase. This extension phase was 4months in duration. Each of the seven patients had an MRI every monthduring the 4 month extension phase. Additionally, each of the sevenpatients was examined neurologically and had serologic studies done atthe end of this phase. No known negative effects 100 milligrams ofprogesterone in combination therapy with 8 milligrams of estrioltreatment were noted.

In closing, it is noted that specific illustrative embodiments of theinvention have been disclosed hereinabove. However, it is to beunderstood that the invention is not limited to these specificembodiments.

Accordingly, the invention is not limited to the precise embodimentsdescribed in detail hereinabove. With respect to the claims, it isapplicant's intention that the claims not be interpreted in accordancewith the sixth paragraph of 35 U.S.C. §112 unless the term “means” isused followed by a functional statement.

While the specification describes particular embodiments of the presentinvention, those of ordinary skill can devise variations of the presentinvention without departing from the inventive concept,

1-39. (canceled)
 40. A method for treating an autoimmune disease in ahuman patient, comprising administering to the patient about 4 mg toabout 16 mg of estriol daily.
 41. The method of claim 40, comprisingadministering to the patient about 8 mg of estriol daily.
 42. The methodof claim 40, wherein the estriol is estriol succinate, estriolsulfamate, or estriol dihexanoate.
 43. The method of claim 40, whereinthe treatment results in a serum estriol concentration of at least about2 ng/mL.
 44. The method of claim 43, wherein the treatment results in aserum estriol concentration of about 10 ng/mL to about 35 ng/mL.
 45. Themethod of claim 40, further comprising administering to the patient aprogesterone.
 46. The method of claim 40, further comprisingadministering to the patient β-interferon, glatiramer acetatecopolymer-1, mitoxantrone, natalizumab, mycophenolate mofetil,paclitaxel, cyclosporine, prednisone, methylprednisone, azathioprine,cyclophosphamide, methotrexate, cladribine, 4-aminopyridine, ortizanidine.
 47. The method of claim 46, comprising administering to thepatient glatiramer acetate copolymer-1, wherein the glatiramer acetatecopolymer-1 is administered at about 0 mg to about 20 mg per day. 48.The method of claim 47, wherein the glatiramer acetate copolymer-1 isadministered at about 20 mg per day.
 49. The method of claim 46,comprising administering to the patient β-interferon, wherein theβ-interferon is interferon β-1a or interferon β-1b.
 50. The method ofclaim 49, comprising administering to the patient interferon β-1a,wherein the interferon β-1a is administered at about 0 μg to about 30 μgonce per week.
 51. The method of claim 50, wherein the interferon β-1ais administered at about 30 μg once per week.
 52. The method of claim49, comprising administering to the patient interferon β-1b, wherein theinterferon β-1b is administered at about 0 mg to about 0.25 mg everyother day.
 53. The method of claim 52, wherein the interferon β-1b isadministered at about 0.25 mg every other day.
 54. The method of claim46, comprising administering to the patient prednisone, wherein theprednisone is administered at about 5 mg to about 60 mg per day.
 55. Themethod of claim 46, comprising administering to the patientmethylprednisone, wherein the methylprednisone is administered at about1 mg to about 2 mg per day.
 56. The method of claim 40, wherein theestriol is administered orally.
 57. The method of claim 40, wherein theautoimmune disease is multiple sclerosis, rheumatoid arthritis,autoimmune thyroiditis, uveitis, myasthenia gravis, Sjogren's syndrome,or Hashimoto's thyroiditis.
 58. The method of claim 57, wherein theautoimmune disease is multiple sclerosis.
 59. The method of claim 58,wherein the autoimmune disease is relapsing remitting multiple sclerosisor secondary progressive multiple sclerosis.
 60. A method for treatingan autoimmune disease in a human patient, comprising: administering tothe patient about 4 mg to about 16 mg of estriol daily; administering tothe patient a progesterone; and administering to the patientβ-interferon, glatiramer acetate copolymer-1, mitoxantrone, natalizumab,mycophenolate mofetil, paclitaxel, cyclosporine, prednisone,methylprednisone, azathioprine, cyclophosphamide, methotrexate,cladribine, 4-aminopyridine, or tizanidine.